Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术(IIF)抗核抗体(ANA)检测法的底物HEp-2细胞,建立抗60kDRo60/SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA,克隆入真核表达载体pEGFP-C1,并转染HEp-2细胞。通过荧光显微镜、免疫印迹法(IBT)及IIF鉴定转染细胞(HEp-Ro60)中Ro60-绿色荧光蛋白(GFP)融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro/SSA对流免疫电泳(CIE)检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达,融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6.7倍(P<0.01),而且2例HEp-2细胞上IIF检测为阴性的血清在HEp-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体,并增加了ANA检出的敏感性。

Application of Indirect Immunofluorescent Test with an Improved HEp-2 Substrate Tranfected with Human Ro60/SSA Autoantigens

To develop an improved substrate for indirect immunofluorescent test(IIF) to detect anti-Ro/SSA autoantibodies,the human 60-kDa Ro/SSA autoantigens(Ro60) cDNAs were obtained from placental cDNA library using PCR and were cloned into the mammalian expression vectorpEGFP-C1.Then the recombinant plasmids were transfected into HEp-2 cells.We confirmed the overexpression,localization and antigenicity of fusion proteins in transfected cells by means of fluorescence microscopy,immunoblotting and IIF.HEp-2 and HEp-Ro60 was analyzed by IIF using a panel of 10 precipitinpositive anti-Ro human sera simultaneously.Stable expression of Ro60-GFP(green fluorescent protein) fusion proteins maintained ten more generations.And Ro60-GFP kept the antigenicity of Ro and had its own characteristic immunofluorescent pattern in HEp-Ro60 cells.The transfectants dramatically increased the sensitivity of IIF testing(a mean increase of 6.7-fold in endpoint titer,P<0.01).Eight(8/10) positive anti-Ro sera showed characteristic immunofluorescent pattern on HEp-Ro60,including two sera which were antinuclear antibodies(ANA) negative on untransfected HEp-2.IIF-ANA in all healthy sera were negative on HEp-Ro60.As a kind of new substrate of IIF,the Ro60 transfectants can be used to detect anti-Ro antibodies.In addition,transfected HEp-2 cells kept the immunofluorescent property of HEp-2 cells in IIF-ANA tests and could be employed as substrate for the routine IIF-ANA detection.The method improved the sensitivity of IIF-ANA.